Enzymatic substrates for multiple detection systems

ABSTRACT

An inventive substrate is provided which includes a substrate compound of formula A-B 1 -B 2 -B 3 -B 4 : wherein A is a sugar moiety; B 1  is a linker moiety allowing the conjugation of moiety A and the remaining structure of the substrate; B 2  is a linker moiety with a free reactive amino group so as to be available for reaction with carboxylic acids or detectable tags; B 3  contains a permanently charged element such as a quaternary ammonium group so as to increase sensitivity for mass spectrometry analysis; and B 4  of various carbon length conferring specificity amongst individual substrates in detection methods. Also provided is a molecule of the formula B 1 -B 2 -B 3 -B 4 , with similar structural characteristics to an enzymatic product produced by the action of a target enzyme on an inventive substrate. Further provided are methods for using inventive substrates for detecting enzymatic activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional, which depends from and claims priorityto U.S. patent application Ser. No. 12/403,790, filed Mar. 13, 2009,which claims priority of U.S. Provisional Patent Application Ser. No.61/036,211 filed Mar. 13, 2008, the contents of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to analytical reagents for detectingenzymatic activity using detection methods such as mass spectrometry,immunoassay, and high performance liquid chromatography. In one aspect,the present invention relates to substrates for detecting lysosomalenzyme activity.

BACKGROUND OF THE INVENTION

Lysosomal storage disorders are a group of inherited disorderscharacterized by deficiencies in specific enzymes in the body, whichresults in the body's inability to break down metabolic substances. Asan example, Fabry disease is a lysosomal storage disorder seen in oneout of every 40,000 people. It is caused by a deficiency in the enzymealpha-galactosidase which results in the body's inability to break downspecific fatty substances called globotriaosylceramides. A secondexample is Gaucher disease, a lysosomal storage disorder caused by aninability to break down fatty substances or lipids calledglucosylceramides (also called glucocerebrosides). Individuals withGaucher disease do not make glucocerebrosidase, an enzyme needed tobreak down these fatty substances. These fatty substances thenaccumulate in cells of the liver, spleen, and bone marrow. A thirdexample is Pompe disease, a lysosomal storage disorder caused by adeficiency in the enzyme acid alpha-glucosidase, which is needed tobreak down certain sugars called glycogen. When the enzyme acidalpha-glucosidase is missing, glycogen accumulates in various tissuesand organs in the body.

Lysosomal storage disorders are, for the most part, childhood disordersalthough some manifest in adulthood. In most of them, patients arenormal at birth and have progressive neurological deteriorationbeginning at some later time. The clinical phenotype depends on the typeand severity of the biochemical defect. Some of these lysosomaldisorders, such as Pompe disease and Krabbe disease, manifest primarilyin infancy. There have been ongoing efforts in developing methods todetect such disorders before the onset of clinical symptoms so thattherapeutic interventions can be initiated.

Over the past decade laboratories that test for metabolic disorders haveintroduced tandem mass spectrometry into their newborn screeningprograms. Tandem mass spectrometry continues to gain popularity in theclinic because this technology allows for assay of many metabolites in asingle sample. For example, this technology has been implemented as aroutine clinical practice for the detection of hereditary metabolicdisorders in newborns using dry blood spot samples (Schulze A et al.,Pediatrics 2003; 111:1399-406). Although lysosomal enzyme activities canbe quantified using tandem mass spectrometry (Gelb M H et al., ClinicalChemistry 50:10, 1785-1796, 2004), published assay methods have not beenreadily adaptable to a clinical setting due to cumbersome procedures andharsh assay components such as chloroform.

A second commonly used clinical assay protocol is the enzyme linkedimmunosorbent assay (ELISA). Several biomarkers are presently used todetect and monitor Gaucher disease (see, for example Aerts, J M, andHollack, C D, Bailhere's Clin. Haematol, 1997; 10:691-709; Deegan P B,et al., Blood Cells Mol Dis, 2005; 35:259-67; Beutler, E, et al., J ExpMed, 1976; 143:975-80). An ELISA for detection of antibodies toaglucerase in which the active component is modified glucocerebrosidasehas also been reported (Richards S M, et al., Blood, 1993; 82:1402;Rosenberg, M. et al, Blood, 1999; 93: 2081-2088). However, these assaysdo not directly detect lysosomal enzyme activity, and instead detectlevels of indirect markers of disease.

Thus, there is a continuing need for improving the methods andcompositions for detecting lysosomal disorders.

SUMMARY OF THE INVENTION

Improved compositions and processes for detecting enzymatic reactionsusing detection systems such as mass spectrometry, immunoassay, and highperformance liquid chromatography are provided according to embodimentsof the present invention.

The invention provides chemical compounds useful for assessing the levelof lysosomal enzyme activity in a sample. Testing of lysosomal enzymeactivity is useful, for example, when screening for metabolic disordersin newborns as well as when assessing an individual having a medicalcondition affecting enzyme activity or one undergoing a medicaltreatment such as enzyme replacement therapy, gene therapy, or bonemarrow transplantation. The chemical compounds described herein includesubstrates for target enzymes and related molecules useful as controlsor standards in enzyme assays.

An inventive substrate has the general formula A-(B¹-B²-B³-B⁴) where Ais a monosaccharide or a disaccharide and B¹ is a C₁-C₂₀ alkyl, C₁-C₂₀having a substituted C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing aheteroatom of N, O or S; B² is an amino acid; 2,6-diaminohexanoic acid;or

where R₁′ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; X is a nullity, oxygen, sulfur, or nitrogen;R₂′ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; C₁-C₂₀ ester; C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl;heteroatom C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing a heteroatom ofN, O or S; Y is a carbon, nitrogen, oxygen, or sulfur nucleophilicgroup; and n is an integer between 1 and 30.

B³ is

where R¹ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; R² is independently in each occurrence a H,a C₁-C₂₀ alkyl, a C₂-C₂₀ alkyl having a substituent of C₁-C₂₀ alkyl; Xis independently in each occurrence a nullity, oxygen, sulfur, ornitrogen; R³ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₆-C₂₀ heterocycliccontaining a heteroatom of N, O or S; n is an integer between 0 and 30,inclusive; R⁴ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₁-C₂₀ carbonyl, C₁-C₂₀amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing aheteroatom of N, O, or S; and B⁴ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ether; C₁-C₂₀ alkyl having a substituent of N, O, or S; C₁-C₂₀ ester;C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl; heteroatom C₆-C₂₀ aryl, C₆-C₂₀heterocyclic containing a heteroatom of N, O or S.

Also provided are compounds of the general formula, which are useful,for example, as controls or standards in enzyme assays:B¹-B²-B³-B⁴  (I)where B¹ is a C₁-C₂₀ alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl,C₆-C₂₀ heterocyclic containing a heteroatom of N, O or S; B² is an aminoacid; 2,6-diaminohexanoic acid; or

where R₁′ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; X is a nullity, oxygen, sulfur, or nitrogen;R₂′ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; C₁-C₂₀ ester; C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl;heteroatom C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing a heteroatom ofN, O or S; Y is a carbon, nitrogen, oxygen, or sulfur nucleophilicgroup; and n is an integer between 1 and 30.

B³ is

where R¹ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; R² is independently in each occurrence a H,a C₁-C₂₀ alkyl, a C₂-C₂₀ alkyl having a substituent of C₁-C₂₀ alkyl; Xis independently in each occurrence a nullity, oxygen, sulfur, ornitrogen; R³ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₆-C₂₀ heterocycliccontaining a heteroatom of N, O or S; n is an integer between 0 and 30,inclusive; R⁴ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₁-C₂₀ carbonyl, C₁-C₂₀amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing aheteroatom of N, O, or S; and B⁴ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ether; C₁-C₂₀ alkyl having a substituent of N, O, or S; C₁-C₂₀ ester;C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl; heteroatom C₆-C₂₀ aryl, C₆-C₂₀heterocyclic containing a heteroatom of N, O or S.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary substrate structure for detecting lysosomalstorage disorders and illustrative method of synthesis.

FIG. 2 is an exemplary substrate structure for detecting lysosomalstorage disorders of general composition and an illustrative method ofsynthesis.

FIG. 3 is an exemplary substrate structure highlighting structuralmoieties.

FIG. 4 is an exemplary substrate structure highlighting multiplefunctional centers amenable to multiple detection methods.

FIG. 5 is a generic enzymatic reaction scheme using an inventivesubstrate.

FIG. 6 is an alternative method of detecting enzymatic activities usingdouble labeling of an inventive substrate.

FIG. 7 is an inventive method of detecting enzymatic reactions usingmass spectrometry that is advantageous in comparison to a prior artreference method.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention has utility as an analytical reagent compositionfor detecting acid hydrolase enzyme activity, such as lysosomal enzymeactivities associated with lysosomal storage disorders. Through theapplication of enzyme substrates and related compounds useful asexperimental controls or standards, that are readily dissolvable insolutions adaptable for analytical methods such as mass spectrometry,HPLC and immunoassay, detecting enzyme activities associated withlysosomal storage diseases is more practical and less cumbersome.

The present invention provides substrates specific for lysosomal enzymesillustratively including acid α-galactosidase A (GLA), acidβ-glucocerebrosidase (ABG), galactocerebroside α-galactosidase (GALC)and acid α-glucosidase (GAA). The action of these enzymes towardinventive substrates is used to measure the corresponding enzymeactivities in a sample, and thus, these substrates can be used to detectlysosomal storage disorders including Fabry (GLA), Gaucher (ABG), Krabbe(GALC) and Pompe (GAA).

An inventive substrate has the general formula of A-(B¹-B²-B³-B⁴) whereA is a monosaccharide or a disaccharide and B¹ is a C₁-C₂₀ alkyl, C₁-C₂₀having a substituted C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing aheteroatom of N, O or S; B² is an amino acid; 2,6-diaminohexanoic acid;or

where R₁′ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; X is a nullity, oxygen, sulfur, or nitrogen;R₂′ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; C₁-C₂₀ ester; C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl;heteroatom C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing a heteroatom ofN, O or S; Y is a carbon, nitrogen, oxygen, or sulfur nucleophilicgroup; and n is an integer between 1 and 30.

B³ is

where R¹ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; R² is independently in each occurrence a H,a C₁-C₂₀ alkyl, a C₂-C₂₀ alkyl having a substituent of C₁-C₂₀ alkyl; Xis independently in each occurrence a nullity, oxygen, sulfur, ornitrogen; R³ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₆-C₂₀ heterocycliccontaining a heteroatom of N, O or S; n is an integer between 0 and 30,inclusive; R⁴ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₁-C₂₀ carbonyl, C₁-C₂₀amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing aheteroatom of N, O, or S; and B⁴ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ether; C₁-C₂₀ alkyl having a substituent of N, O, or S; C₁-C₂₀ ester;C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl; heteroatom C₆-C₂₀ aryl, C₆-C₂₀heterocyclic containing a heteroatom of N, O or S.

Specificity of the inventive substrate for a particular lysosomal enzymeis provided in part by structural variations in the sugar moiety A suchas A being a monosaccharide or a disaccharide. Exemplary sugar moietiesinclude α-D-Glucose for detecting Pompe disease; β-D-Glucose fordetecting Gaucher disease; α-D-Galactose for detecting Fabry disease;and β-D-Galactose for detecting Krabbe disease.

B¹ is a linker moiety which functions to allow conjugation of the sugarmoiety A to the remaining structure of the substrate. B¹ also functionsas a spacer between the sugar moiety A and the remaining structure ofthe substrate so as to provide flexible access for a target enzyme. Thelinker arm B¹ can be designed so as to confer relatively hydrophiliccharacteristics. Particularly, the linker arm B¹ can have a hydrophenolstructure. Thus, an inventive substrate of the general formula ofA-B¹-B²-B³-B⁴ can be hydrophilic in a solvent such as pure methanol orpure ethanol. The B¹-B²-B³-B⁴ moiety in toto generally is hydrophilic.As such, the inventive substrates can be soluble in aqueous buffersystems.

B² provides a nucleophilic group(s) for interactions with a solidsupport or detectable tag, such as a fluorescent tag. Preferably, B² isa derivative of an alkyl diamino acid, such as 2,6-diaminohexanoic acid.In the case of 2,6-diaminohexamoic acid, the 6-amino group optionallyserves as a nucleophile for binding to a solid support or detectabletag.

A quaternary ammonium group is a component of B³. Upon an enzymaticreaction, a cleaved product of B¹-B²-B³-B⁴ carries with it a permanentpositive charge located on the B³. This property results in robustsignal in tandem mass spectrometry analysis. Additionally, the permanentcharge makes the inventive substrate more soluble in aqueous solutionsso as to avoid the need for using solvents such as chloroform. Incomparison to previously described substrates, a substrate according tothe present invention is generally more hydrophilic and requires less orno need for detergents. This results in simplified assay proceduresbecause, like the use of chloroform, the use of detergents can requirecumbersome clean up steps including the labor-intensive liquid-liquidand solid phase extractions.

An inventive substrate is structurally terminated by a B⁴ group. B⁴ canbe structurally tailored to provide different chain lengths. Suchdifferent chain lengths are useful for distinguishing differentsubstrates, as well as enzymatic products thereof, from each other inenzyme assays. For example, in mass spectrometry, a substrate containinga 13 carbon atom chain has a different mass-to-charge ratio than asubstrate containing a 14 carbon atom chain and as such, substratescontaining 13 carbon atom or 14 carbon atom chains can be distinguished.Similarly, in immunoassay formats, substrates having differing chainlengths can be distinguished using antibodies selective for particularchemical moieties.

In one embodiment, the invention provides a reagent of formulaA-B¹-B²-B³-B⁴ wherein A is a monosaccharide or a disaccharide andpreferably an aldohexose or a ketohexose; B¹ is a phenol, a nitrophenol,or a phenyl ester such as a phenyl benzoate; B² contains an alkyl groupsuch as the amino acid lysine; B³ contains a pendent quaternary ammoniumcation extending from structure that prior to condensation to B¹ aloneor also with a B⁴ tail is carnitine.

The present invention provides methods for detecting enzyme activity.The activity of a particular enzyme can be assessed by its capability orrate of acting on a cognate substrate to product enzymatic products. Inthe case of an inventive substrate, action of a target enzyme onsubstrate A-B¹-B²-B³-B⁴ results in generation of two products: A andB¹-B²-B³-B⁴. By determining the amount of an enzymatic product, such asB¹-B²-B³-B⁴, in a sample, the activity of the target enzyme can bedetermined. For applications in which a quantitative assessment ofB¹-B²-B³-B⁴ amount is desired, a known amount of an internal standardcorresponding to B¹-B²-B³-B⁴, as is described in more detail below, canbe included in the sample. As such, the invention provides compoundshaving the general formula of B¹-B²-B³-B⁴.

The activities of certain lysosomal enzymes in the blood of anindividual can be used to test whether that individual has a lysosomalstorage disorder. Therefore, the invention provides substrates fordetecting medical conditions, in particular, lysosomal storage disorderssuch as Pompe disease, Gaucher disease, Fabry disease and Krabbedisease. For detecting Pompe disease, an exemplary sugar moiety isα-D-glucose and an exemplary B¹-B²-B³-B⁴ portion is4-aminophenyl-diaminohexanoyl-cartnitinyl-alkyl chain with B⁴ of 10-20carbons. For detecting Gaucher disease, an exemplary sugar moiety isβ-D-glucose and an exemplary B¹-B²-B³-B⁴ portion is4-aminophenyl-diaminohexanoyl-carnitinyl-alkyl with B⁴ of 10-20 carbonsin length. For detecting Fabry disease, an exemplary sugar moiety isα-D-galactose and an exemplary B¹-B²-B³-B⁴ portion is4-aminophenyl-diaminohexanoyl-carnitinyl-alkyl with B⁴ of 10-20 carbonsin length. For detecting Krabbe disease, an exemplary sugar moiety isβ-D-galactose and an exemplary B⁴ portion is4-aminophenyl-diaminohexanoyl-carnitinyl-alkyl with B⁴ of 10-20 carbonsin length. A specific example of an inventive substrate specific fordetecting Krabbe disease has a group A of β-D-Galactose, a group B¹ of amethylene, a group B² of an amidylaminoacyl group, a group B³ of aamidyl terminating with a quaternary ammonium, and group B⁴ of alkenylalcohol with a carbon length of 12 to 20. A specific example of aninventive substrate for detecting Gaucher disease has a group A ofβ-D-Glucose, a group B¹ of a methylene, a group B² of an amidylaminoacylgroup, a group B³ of a amidyl terminating with a quaternary ammonium,and group B⁴ of alkenyl alcohol with a carbon length of 12 to 20.

An inventive substrate can be tailored for assaying a variety ofenzymes, in particular, enzymes associated with a disease state or birthdetect, or one otherwise useful for medical purposes. Such tailoring ispossible because a variety of monosaccharide and disaccharide groups canbe present at A of the general formula A-B¹-B²-B³-B⁴. Even for a newlyidentified target enzyme, once its specificity for monosaccharide and/ordisaccharide groups is determined using routine methods, an inventivesubstrate can be readily prepared using guidance provided herein.Non-limiting examples of enzymes which can be assayed using an inventivesubstrate as described herein include acid α-galactosidase A, acidβ-glucocerebrosidase, acid galactocerebroside α-galactosidase, acidsphingomyelinase, and acid α-glucosidase

As it is envisioned in the present invention, one can synthesizesubstrates with different sugars, each specific to a particularlysosomal enzyme, and each having a different chain length in subgroupB² or B⁴. This inventive system provides for optional multiplex assayswhere two or more lysosomal enzymes are analyzed in the same sample orsample receptacle using structurally similar yet enzyme specificsubstrates.

The present invention provides compounds that function as experimentalcontrols or standards useful for assessing the amount of enzymaticproduct in a sample or sample receptacle. For use in mass spectrometrymethods, an internal standard corresponding to a particular inventivesubstrate is structurally identical to its enzymatic product, exceptthat the internal standard differs in mass-to-charge (m/z) ratio. Thus,the internal standards of the present invention include modified formsof enzymatic products, for example, stable isotope-labeled analogs ofenzymatic products in which one or more atoms are replaced bycorresponding atomic isotopes so as to create a shift in the mass. Whenthe internal standard and enzymatic product are analyzed by massspectrometry, the resulting spectrum reveals a spatial separation of theinternal standard and enzymatic product, each represented by its ownpeak. The known amount of internal standard is reflected by peakmagnitude at its known m/z ratio. The amount of enzymatic product can beassessed by comparison of peak magnitude at its known m/z, relative tothe peak magnitude of the internal standard. An example of isotopiclabeling to produce an internal standard is the replacement of ¹H on anacyl group of B⁴ with ²D. As a result, a “heavier” internal standardmolecule with the substituted ²D has a different m/z from the enzymaticproduct, as detected on a mass spectrum.

In a particular embodiment, an internal standard is labeled withdeuterium to cause a mass change of 3 to 9 Daltons from thecorresponding cleaved product. In another particular embodiment, acombined B²-B³-B⁴ subgroup is a lysine-acylcarnitine with a positivelycharged quaternary ammonium moiety and the acyl tail is of carbon lengthfrom 12 to 18.

In one embodiment the inventive substrates are labeled with a detectabletag. Many fluorescent probes are recognized in the art as useful forlabeling reactive amines. A particularly sensitive target for specificlabeling of biomolecules is the side chain amino group of lysine. Apreferred embodiment of the inventive substrates includes a lysineresidue at position B² that possess this active terminal amino group.Illustrative examples of detectable tags suitable for labeling theinventive substrates include fluorophores such as isothiocyanates,dansyl and other sulfonyl chlorides, 7-nitrobenz-2-oxa-1,3-diazolederivatives, fluorescamine, and the like.

An inventive substrate can be used in a variety of physical formats, forexample, in solution as well as linked or immobilized to solid supports.A solid support can be composed of a natural or synthetic material, anorganic or inorganic material, such as a polymer, resin, metal or glass,and combinations thereof. A suitable solid support can have a variety ofphysical formats, which can include for example, a membrane; column; ahollow, solid, semi-solid, pore or cavity-containing particle such as abead; a gel; a fiber, including a fiber optic material; a matrix andsample receptacle. Non-limiting examples of sample receptacles includesample wells, tubes, capillaries, vials and any other vessel, groove orindentation capable of holding a sample. A sample receptacle can becontained on a multi-sample platform, such as a microplate, slide,microfluidics device, and the like. Many suitable particles are known inthe art and illustratively include Luminex®-type encoded particles,encoded fiber optic particles, magnetic particles, and glass particles.Covalent interaction of an inventive substrate and/or enzymatic cleavageproduct thereof with a solid support is useful for retaining thesubstrate and/or product during washing procedures performed in someassay formats, thus, producing a robust and accurate signal of enzymaticactivity.

When use of a solid support is desired for an assay format, the presenceof the exemplary lysine group B² can be used, for example, for covalentbonding to high-binding solid supports. High binding solid supports aresurfaces having exposed moieties that are chemically active or otherwisecapable of covalent or high affinity binding to an inventive substrateor internal standard. As an example, Corning Life Sciences produceshigh-binding microwell plates that are irradiated to break the benzenering and produce exposed carboxylic acids. These carboxylic acids areamenable to nucleophilic attack such as by the terminal amino group onthe lysine derivative component of a preferred embodiment substrate.This reaction is rapid and produces a tight interaction between thesubstrate/product and the high-binding surface.

The methods described herein can be performed in a multiplexed formatsuch that a plurality of samples are assayed simultaneously. Anillustrative multiplexed format involves using physically and/orchemically coded particles. Use of coded particles in multiplexedformats has been described, for example, in U.S. Pat. No. 6,649,414 andU.S. Pat. No. 6,939,720. Because the codes allow particles to bedistinguished from each other, a plurality of distinct particles can bepresent in a single reaction mixture, allowing a plurality of differentsamples or different enzymes to be assayed simultaneously. Codes onparticles can correspond, for example, to sample origins, particularenzymes to be assayed, particular substrates present, and the like,depending on the experimental goal of the user.

A sample useful in the methods of the invention contains or is suspectedof containing one or more target enzymes. Target enzymes can becontained in samples obtained from an individual, as well as fromlaboratory materials, such as cell lines, and synthetic protein sources.Exemplary sample sources include tissue homogenates; cell culturelysates; biological fluids including urine, blood in liquid or dry form,tears, saliva, and cerebrospinal fluid. A sample can be furtherfractionated, if desired, to a fraction containing particular celltypes. For example, a blood sample can be fractionated into serum orinto fractions containing particular types of blood cells such as redblood cells or white blood cells (leukocytes). If desired, a sample canbe a combination of samples from a subject such as a combination of atissue and fluid sample, and the like. In a specific embodiment, thesample is blood, which can be, for example, whole blood or a bloodfraction thereof, or reconstituted from a dry blood sample.

Methods for obtaining samples that preserve the activity or integrity ofmolecules in the sample are well known to those skilled in the art. Suchmethods include the use of appropriate buffers and/or inhibitors,including nuclease, protease and phosphatase inhibitors, which preserveor minimize changes in the molecules in the sample. Such inhibitorsinclude, for example, chelators such as ethylenediamine tetraacetic acid(EDTA), ethylene glycol bis(P-aminoethyl ether)N,N,N1,N1-tetraaceticacid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride(PMSF), aprotinin, leupeptin, antipain and the like, and phosphataseinhibitors such as phosphate, sodium fluoride, vanadate and the like.Appropriate buffers and conditions for isolating molecules are wellknown to those skilled in the art and can be varied depending, forexample, on the type of molecule in the sample to be characterized (see,for example, Ausubel et al. Current Protocols in Molecular Biology(Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane,Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press(1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, ColdSpring Harbor Press (1999); Tietz Textbook of Clinical Chemistry, 3rded. Burtis and Ashwood, eds. W.B. Saunders, Philadelphia, (1999)). Asample also can be processed to eliminate or minimize the presence ofinterfering substances.

Samples in the form of a dry blood spot are commonly used when screeningblood from newborns and children patients. To prepare these samples,blood is collected and retained on a filter paper. For analysis, thedried blood is eluted from the filter paper into an aqueous solution,which generally contains a buffer such as phosphate buffer saline and aprotease inhibitor. Specific examples of protease inhibitor conditionsinclude for example, include one or more of the following: AEBSFhydrochloride in a final concentration of 50 to 400 μg/ml, EDTA disodiumdehydrate in a final concentration of 0.2 to 25 mg/ml, leupeptinhemisulfate in a final concentration of 0.5 to 1 μg/ml, and pepstatin Ain a final concentration of 0.5 to 1 μg/ml. The use of a universal assaysolution to extract a single dry blood sample, or other type of sample,for subsequent distribution into multiple assay reactions can be usedfor automatic and high throughput screening. A single extraction of adry sample avoids the need to obtain several sample punches from thesame sample, or to collect aliquots of other sample sources andaccordingly reduces variation caused by inhomogeneous distribution ofblood on the filter paper and errors in sample transfer. When using drysamples, extraction efficiency may vary with the different enzymes beinganalyzed. In these and other types of samples, the target enzymes mayhave different levels of activities when contained in different assaysolutions. Composition of the inventive universal assay solution isoptionally chosen such that each enzyme to be tested is active.

The inventive substrates and products can be used in a variety of assayformats. The substrate can be detected in an assay when it is desired toobserve substrate consumption during an enzymatic assay, while theproduct can be detected in the assay when it is desired to observe itsformation during an enzymatic assay. Both substrate and product can bedetected when it is desired to observe the enzymatic reaction from bothperspectives, for example, to confirm that the amount of productproduced correlates with the amount of substrate consumed.

For example, the amount of substrate A-B¹-B²-B³-B⁴ or productB¹-B²-B³-B⁴ can be detected using established tandem mass spectrometryprocedures. An exemplary enzyme assay employing mass spectrometry can beperformed as follows. A sample is incubated with an inventive substratefor a time period that allows formation of an enzymatic product. Duringthe incubation period, the substrate is cleaved by a target enzymepresent in a blood sample to form a respective B1-B2-B3-B4 product. Thereaction is then quenched by adding a reagent that precipitates proteincomponents. Exemplary reagents include alcohol, acetonitrile and dilutetrifluoro acetic acid. A portion of the incubation mixture is thentransferred to a new assay vessel. Optionally, a dilution reagent suchas methanol, acetonitrile, water-methanol mixtures or water-acetonitrilecan be added to dilute the transferred portion. The sample so dilutedreduces the amount of endogenous competing material so as to relativelyincrease the sensitivity of the tandem mass spectrometry analysis. Othertypes of reagents are selected by those skilled in the art to becompatible with tandem mass spectrometry analysis.

The diluted sample is directly injected into the tandem massspectrometer either manually or automatically with the aid ofautosamplers and liquid handlers. If desired, the sample can bederivatized prior to analysis. Reagents are selected to be non-hostileto the MS/MS system. For example, suitable solvents lack detergents andcorrosive agents, such as chloroform. Pure ethanol and pure methanol areoften used simply because they easily vaporized upon mechanical dryingprocesses.

The tandem mass spectrometer can be set to simultaneously detect theadded substrate, the corresponding resulting enzymatic product and thecorresponding internal standards. Such detection is accomplished bymeans of parent ion scans, precursor ion scans or multiple reactionmonitoring scans.

The amount of substrate A-B¹-B²-B³-B⁴ or product B¹-B²-B³-B⁴ formedduring an enzymatic assay also can be detected using antibodies andother target-specific binding molecules. For immunoassays, an antibodycan be used to detect the substrate, product or both. Antibodies usefulin such methods can be specific, such that they recognize individualsubstrates, or non-specific, such that they recognize many or allsubstrates. An illustrative example is an antibody generated to thecombination B1-B2 nitrophenol-lysine.

The antibody is illustratively produced in animals including mouse, rat,rabbit, horse, donkey, or other suitable animal used for the productionof antibodies. In some applications, it is useful to label an antibodywith a detectable tag, such as a fluorescent tag. When using anunlabeled antibody, detection can be performed by using a secondaryantibody that is specific for the species IgG of the primary antibody islabeled illustratively with a fluorescent marker such as rhodamine. Itis appreciated in the art that other antibody detection systems aresimilarly operable in the instant invention such as horseradishperoxidase labeled antibodies, or alkaline phosphatase labeledantibodies.

When testing multiple enzymes in a single sample by providing multipleenzyme-specific substrates, antibodies that recognize and distinguishbetween the substrates, or products thereof, and be used. Complexes ofantibodies bound to enzyme-specific substrates, or products thereof, canbe distinguished from each other using many methods. In one scenario,samples containing target enzymes are contacted with substrates linkedto particles in an assay solution. In this example, each particle islinked to a particular substrate, and there are multiple particlesrepresenting each substrate. The target enzymes act on the substrates toproduce products A and B¹-B²-B³-B⁴. The B¹-B²-B³-B⁴ product remainsbound to the particle, while the A product is released into solution.Antibodies that recognize specific B¹-B²-B³-B⁴ products are thencontacted with the assay solution. The antibodies will bind to theproducts, if produced during the enzymatic assay, to produce particleshaving bound antibodies. To distinguish different products contained onthe particles, antibodies having different product specificities canhave different detectable moieties, such as different fluorescent tags.As an alternative to detecting enzymatic products, antibodies thatrecognize substrate A-B¹-B²-B³-B⁴ can be used to detect substrateremaining on the beads after incubation with enzymes. In this situation,either product A or B¹-B²-B³-B⁴ would remain attached to the bead, if anenzymatic reaction occurred. In either case the selected substratespecific antibody would not significantly cross-react with productattached to the bead.

In another scenario, samples containing target enzymes are contactedwith substrates linked to encoded particles in an assay solution. Theencoded particles have a feature, such as a bar code or optical profile,which allows them to be distinguished from each other. For example,encoded particles can have different bar codes corresponding todifferent target enzyme substrates. In the assay, the target enzymes acton the substrates to produce products A and B¹-B²-B³-B⁴. The B¹-B²-B³-B⁴would remain bound to the particle, while the A product would bereleased into solution, or visa versa. Antibodies that recognizespecific products are then contacted with the assay solution. Becausethe encoding of the particle indicates which substrate is attached tothe particle, antibodies need not be specific for particular products,and thus one type of antibody can be used to detect products derivedfrom multiple different substrates. Such non-specific antibodies willbind to the products, if produced during the enzymatic assay, to produceparticles having bound antibodies. Particles having bound antibodies arethen distinguished from those without antibodies, for example, bydetecting a tag on the antibodies or physical behavior of the particles.The different products contained on the antibody-bound particles can bedetermined based on the encoding of each particle.

As another example of an immunoassay format, antibodies directed toparticular substrates are generated. Following quenching of an enzymaticreaction, the reaction solution is transferred to a high-bindingmicrotiter plate whereby the reactive B² moiety covalently attaches tothe plate via a terminal amino group. The enzyme and assay solutioncomponents are removed by washing. The specific primarily antibody isthen incubated in each assay well followed by subsequent washing toremove unbound antibody. A secondary antibody is optionally used fordetection and quantitation. The more product formed per unit time ofinitial reaction the greater the activity of the measured enzyme.

In an alternative immunoassay format, an antibody specific for the B¹-B²subgroup is optionally used as a capture antibody on the surface of themicrotiter plate in a standard sandwich ELISA assay. A primary antibodywith a unique epitope on the product such as one directed to the B⁴moiety (or the B⁴ moiety is modified with a specific binding pair membersuch as biotin) is used for detection. As is recognized in the art, alabeled secondary antibody is optionally used for detection as describedabove.

In an additional immunoassay format, an exemplary antibody reacts withthe α-D-glucose A group bound to the inventive hydrophenol B¹ moiety.The substrate is attached to a solid support using a lysine B² moiety.Alternatively, the substrate is provided in solution, the reaction istransferred to a sample receptacle, in which Following quenching of anexemplary enzymatic reaction, the reaction solution is transferred to ahigh-binding microtiter plate whereby the reactive B² moiety covalentlyattaches to the receptacle via a terminal amino group. As anotheralternative, a capture antibody specific for an alternate epitope on theinventive product/substrate is employed. The unreacted enzyme and buffercomponents are removed by washing. The antibody specific to the A-B¹-B²moiety is then incubated in each assay well for detection andquantitation of remaining substrate. The greater the substrate remainingafter the initial enzyme reaction, the lower the activity of the enzyme.

The antibody is illustratively unlabeled and produced in animalsincluding mouse, rat, rabbit, horse, donkey, or other suitable animalused for the production of antibodies. A secondary antibody that isspecific for the species IgG of the primary antibody is labeledillustratively with a fluorescent marker such as rhodamine andsubsequently used for detection of remaining substrate. It isappreciated in the art that other antibody detection systems aresimilarly operable in the instant invention such as horseradishperoxidase labeled antibodies, or alkaline phosphatase labeledantibodies.

In another example of a suitable immunoassay format, monoclonal mouseantibody specific for the exemplary the α-D-glucose A group bound to theinventive hydrophenol B¹ moiety and lysine B² moiety is itself labeledillustratively by a fluorescent marker. In this system multiplelysosomal enzymes are optionally simultaneously analyzed for activitytoward a variety of specific substrates. An illustrative exampleincludes a two enzyme system wherein inventive substrate are employed,one specific for GLA and another specific for GAA. Each substrate issimultaneously added to the reaction with the biological sample. As eachsubstrate optionally contains a lysine B² group, both will similarlybind to the high-binding microtiter plate. Two antibodies, each specificfor its respective inventive substrate are added to the microtiter platefollowing washing as above. Each antibody is illustratively labeled witha different fluorophore such as rhodamine or cyanine. As such thebinding of each antibody is detected and quantitated withoutinterference from the other, and the amount of each enzyme activity isdetectable in the same well of the microtiter plate from the samesample.

Another illustrative assay format is performed using mass spectrometry.An assay for target enzymes is performed by first obtaining a sampleillustratively including serum, plasma, whole blood, urea, saliva, otherbiological fluids or tissue lysates, recombinant or native purifiedenzyme in solution, or chemically or functionally modified enzyme inbiological fluid or liquid medium. A portion of the filter paper sampleis then excised and deposited in a non-binding assay tube or micro titerplate well to which an assay solution is added. The assay solutioncomprises aqueous buffers, a substrate, a standard, as well as proteaseinhibitors. The sample mixture is then incubated for a determined periodof time in the range of 30 minutes to 20 hours at a particulartemperature ranging from 30 to 41° C. Once incubation is complete, theenzymatic reaction is terminated by addition of a stopping solution. Astopping solution is illustratively 0.4 M glycine/NaOH pH 10.4 added at6× reaction volume. Leonard R, et al., J. Biol. Chem., 2006;281:4867-75; Boot, R G, et al., J. Biol. Chem., 2006; 282:1305-12. Theamount of product formation is determined by transferring a known volumeof sample to a high-binding assay tube or microtiter plate and incubatedfor 5 minutes to 2 hours. The unbound material is removed by washing.Detection of the intact substrates or products is illustrativelyperformed using a coupled peroxidase enzyme approach. In a furtherscenario, the level of released glucose or galactose product is measuredin real time by a coupled enzyme approach. A non-limiting exampleinvolves the release of glucose from an inventive substrate specific foralpha-glucosidase in diagnosis of Pompe disease. In this assay methodglucose is reacted with glucose oxidase producing glucolactone andreleasing hydrogen peroxide. The released hydrogen peroxide is detectedby reaction with peroxidase to produce a fluorescent molecule that ismeasured on a standard fluorometer. Examples of suitable peroxidases arehorseradish peroxidase or any other peroxidase known in the art. Thehydrogen peroxide released by glucose oxidase interacts with a detectorsubstrate molecule. The peroxidase catalyzes conversion of thissubstrate to a fluorescent product. A detector molecule suitable for usewith the inventive substrates includes Amplex Red that is oxidized in toproduce the fluorescent product resorufin. Amplex Red and kits fordetecting free glucose are available from Invitrogen Corp. The increasein red fluorescent product is detected on a fluorometer set with anexcitation wavelength at 571 and an emission wavelength at 585 with theband pass set at 5 nm. The greater amount of glycosidase activity themore rapidly the red fluorescent product is produced.

In a preferred embodiment multiple substrates for different lysosomalenzymes are generated with unique B¹-B⁴ structure. This prevents productinhibition of one enzyme that is particularly important should thecatalytic activity of one enzyme toward one inventive substrate be muchgreater than the catalytic activity of the other enzyme for itscorresponding inventive substrate. This is additionally important inconditions where a single mutant glycosidase is being screened in apanel of substrates for 6 or more lysosomal enzymes. The product formedby the other lysosomal enzymes may inhibit the function of the loweractivity enzyme such that its activity is not accurately measured. Thus,the specificity of the substrate and the product for each enzyme isappreciated to be optionally distinct.

When more than one enzyme is detected simultaneously by combiningmultiple substrates directed to respective enzymes, the substrates maydiffer not only in the type of sugar moiety which confers enzymespecificity, but also in the length of the B⁴ tail moiety. This isparticularly important with the use of MS/MS as a detection tool sincethe differentiated inventive substrate molecules having correspondingdifferentiated mass index correspond to various enzymes being examined.

The approach described for assaying enzymes using substrate reagents,immunoassay, HPLC, and mass spectrometry according to the presentinvention may be broadly applied. The multiplex techniques may beexpanded to assay dozens or more enzymes simultaneously in a singlereaction, obviating the need for multiple assays to assist in confirmingdiagnoses of rare disorders. The method may be used to measure severalenzymes simultaneously when evaluating the rate of chemical flux througha specific biochemical pathway or for monitoring biochemical signalingpathways. Because of the high sensitivity of the ESI-MS detectionemployed, which requires only sub-microgram quantities of the substratereagents per assay, the synthesis of several hundred substrate reagentson a low-gram scale becomes practical and economical.

In a preferred embodiment two lysosomal enzymes are simultaneouslymeasured for activity by the use of inventive substrates. In analternative embodiment 2-6 lysosomal enzymes are simultaneouslymeasured.

As another exemplary format for use of the inventive substrates, thesubstrates can be labeled with the same fluorophore, but possesssignificant mass or charge characteristics that differentiate one fromthe other. The amount of product produced following an enzymaticcleavage reaction is detected by reversed phase high performance liquidchoromatography (HPLC). Reactions are quenched by the addition ofalcohol, acetonitrile or dilute trifluoro acetic acid. A portion of theincubation mixture is transferred to a new assay vessel to which isadded a neat solution such as methanol, acetonitrile, water-methanolmixtures or water-acetonitrile. The reaction products and unreactedsubstrate are separated on a 5 μm particle size C₁₈HPLC column anddetected by a fluorescent detector or set of detectors. The amount ofproduct is calculated based on a standard curve generated usingincreasing amounts of the relevant product.

It is appreciated in the art that multiple substrates for multipleenzymes are optionally simultaneously detected by the HPLC method. Ifsubstrates with sufficiently different mass or retention characteristicsare used each product is resolvable in the HPLC column and can bequantified in a single assay. Alternatively, each substrate is labeledwith a different fluorophore that has different or the same excitationor emission properties. Detection may be by a family of fluorescentdetectors that can simultaneously quantify individual products from eachother and their corresponding labeled substrate. Other methods ofdetection are similarly suitable and are known in the art.

FIG. 1 shows α-D-glucose, modified with a 4-aminophenyl group is reactedwith a di-protected lysine group. Methods for di-protecting lysine areknown in the art and illustrative examples are described in WO 01/27074.The intermediate A-B¹-B² is formed which is specifically deprotected atthe alpha-amino group to provide a suitable reactive site for subsequentsynthetic steps. Methods of site-specific deprotection are known in theart. The alkyl chain is then added. An exemplary alkyl chain is providedby dodecyl-carnitine (B³-B⁴) which is synthetically added to the A-B¹-B²intermediate to form a full length substrate molecule (A-B¹-B²-B³-B⁴).This substrate molecule is active as a GAA specific substrate and isoptionally used in analyses involving mass spectrometry or othersuitable assay and detection method. In another preferred embodiment,the 6-amino group on B² is deprotected so as to produce a similarlyreactive substrate. It is appreciated in the art that synthesis ofnumerous derivatives of the above illustrated substrate are similarlyachieved such as by varying the carbon chain on the acylcarnitine,altering the sugar A group or the B¹ group. Also, numerous derivativesof lysine (B²) or other alkyl groups are similarly suitable.

It is appreciated in the art that similar synthetic pathways aresuitable for production of the inventive substrates. FIG. 2 depicts asynthetic pathway similar to FIG. 1 except for the protective groups andthe choice of acylcarnitine whereby n is a value between 1 and 30.Preferably, n is between 1 and 20.

FIG. 3 depicts an exemplary substrate structure for detecting lysosomalstorage diseases. The structure is composed of a sugar (A) in the formof a glucose or a galactose and an aliphatic group B. Group B is furthercomposed of a linker arm (B¹) in the form of a nitrophenyl, a B²subgroup of a lysine, a B³ subgroup of a carnitinyl, and a B⁴ subgroupin the form of an alkyl with carbon length in the range of 10 to 30. Aquaternary ammonium cation located on the B³ subgroup avoids the need offurther ionization otherwise needed for mass spectrometry detection.

FIG. 4 depicts the multiple functional groups that provide multipleassay format detection of enzymatic activity. The terminal amino groupon B² is capable of forming covalent bonds with carboxylic acids onsolid supports. The presence of the quaternary ammonium on B³ provides apermanently charged moiety such that subsequent ionization is notrequired for analysis by MS/MS providing a robust and easily detectableand quantifiable detection of lysosomal enzyme activity.

FIG. 5 demonstrates a generic enzymatic reaction using an inventivesubstrate. Upon specific affinity binding and enzymatic reaction, thesubstrate is cleaved into two groups, a sugar moiety A and an aliphaticgroup B. The group B is optionally composed of a nitrophenyl, a lysine,a carnitinyl, and long-chain alkyl moieties. Both groups are optionallythen analyzed by MS/MS. An internal standard is also concurrentlysubject to the MS/MS analysis. The internal standard is an isotopicallylabeled analog of B with deuterium to replace hydrogen atom(s) on amethyl group. Alternatively, the deprotected terminal amino group on B²is reactive with a solid support such that the presence or absence ofproduct is optionally detected using immunoassay methods. Thedeprotected terminal amine can also be derivatized with a fluorescentreagent for HPLC, or other suitable detection method.

FIG. 6 illustrates exemplary inventive methods depicting detection ofenzymatic reactions using mass spectrometry that are advantageous incomparison to prior art methods.

In the prior art, lysosomal enzyme substrates each require a uniquesynthetic pathway. In contrast, the inventive substrates can besynthesized using a common synthetic pathway. Having a common syntheticpathway for two or more of the inventive substrates means significantsavings in production environments due to shorter and less complexproduction processes and the use of common raw materials. The inventivesubstrates also can be rapidly synthesized relative to the prior art.Synthesis is accomplished in as little as three steps. Synthesis of apreferred inventive substrate with specificity for the lysosomal enzymeGAA (Pompe disease) is illustrated in FIG. 1.

It is further appreciated that the compound B¹-B²-B³-B⁴ is alsofunctional as an antagonist, an analytical control, or for clinicaltreatment of disease such as hypothyroidism, diabetes, and HIV. TheB¹-B²-B³-B⁴ molecule is: B¹ is a C₁-C₂₀ alkyl, C₁-C₂₀ having asubstituted C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing a heteroatom ofN, O or S; B² is an amino acid; 2,6-diaminohexanoic acid; or

where R¹′ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; X is a nullity, oxygen, sulfur, or nitrogen;R₂′ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; C₁-C₂₀ ester; C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl;heteroatom C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing a heteroatom ofN, O or S; Y is a carbon, nitrogen, oxygen, or sulfur nucleophilicgroup; and n is an integer between 1 and 30.

B³ is

where R¹ is a C₁-C₂₀ alkyl; C₄-C₂₀ ether; C₁-C₂₀ alkyl having asubstituent of N, O, or S; heteroatom C₆-C₂₀ aryl, C₁-C₂₀ carbonyl,C₁-C₂₀ amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containinga heteroatom of N, O, or S; R² is independently in each occurrence a H,a C₁-C₂₀ alkyl, a C₂-C₂₀ alkyl having a substituent of C₁-C₂₀ alkyl; Xis independently in each occurrence a nullity, oxygen, sulfur, ornitrogen; R³ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₆-C₂₀ heterocycliccontaining a heteroatom of N, O or S; n is an integer between 0 and 30,inclusive; R⁴ is independently in each occurrence a nullity, C₁-C₂₀alkyl, C₁-C₂₀ having a substituted C₆-C₂₀ aryl, C₁-C₂₀ carbonyl, C₁-C₂₀amidyl, C₁-C₂₀ ether, C₆-C₂₀ aryl, C₆-C₂₀ heterocyclic containing aheteroatom of N, O, or S; and B⁴ is a nullity or C₁-C₂₀ alkyl, C₄-C₂₀ether; C₁-C₂₀ alkyl having a substituent of N, O, or S; C₁-C₂₀ ester;C₁-C₂₀ alcohol; C₁-C₂₀ alkenyl; heteroatom C₆-C₂₀ aryl, C₆-C₂₀heterocyclic containing a heteroatom of N, O or S.

In an alternative embodiment the inventive substrates A-B¹-B²-B³-B⁴ areoptionally synthesized with a non-hydrolyzable link between A and B¹.This produces suicide substrates that maintain high specificity fortheir target lysosomal enzyme whereby specificity is conferred by boththe A and B⁴ moieties. These molecules serve as more specific and potentinhibitors of enzyme function.

FIG. 1 shows α-D-glucose, modified with a 4-aminophenyl group is reactedwith a differentially protected lysine group. Methods for differentiallyprotecting lysine are known in the art and illustrative examples aredescribed in WO 01/27074. The intermediate A-B¹-B² is formed which isspecifically deprotected at the alpha-amino group to provide a suitablereactive site for subsequent synthetic steps. Methods of site-specificdeprotection are known in the art. The alkyl chain is then added. Anexemplary alkyl chain is provided by dodecyl-carnitine (B³-B⁴) which issynthetically added to the A-B¹-B² intermediate to form a full lengthsubstrate molecule (A-B¹-B²-B³-B⁴). This substrate molecule is active asa GAA specific substrate and is optionally used in analyses involvingmass spectrometry or other suitable assay and detection method. Inanother preferred embodiment, the 6-amino group on B² is deprotected soas to produce a similarly reactive substrate. It is appreciated in theart that synthesis of numerous derivatives of the above illustratedsubstrate are similarly achieved such as by varying the carbon chain onthe acylcarnitine, altering the sugar A group or the B¹ group. Also,numerous derivatives of lysine (B²) or other alkyl groups are similarlysuitable.

It is appreciated in the art that similar synthetic pathways aresuitable for production of the inventive substrates. FIG. 2 depicts asynthetic pathway similar to FIG. 1 except for the protective groups andthe choice of acylcarnitine whereby n is a value between 1 and 30.Preferably, n is between 1 and 20.

FIG. 3 depicts an exemplary substrate structure for detecting lysosomalstorage diseases. The structure is composed of a sugar (A) in the formof a glucose or a galactose and an aliphatic group B. Group B is furthercomposed of a linker arm (B¹) in the form of a nitrophenyl, a B²subgroup of a lysine, a B³ subgroup of a carnitinyl, and a B⁴ subgroupin the form of an alkyl with carbon length in the range of 10 to 30. Aquaternary ammonium cation located on the B³ subgroup avoids the need offurther ionization otherwise needed for mass spectrometry detection.

FIG. 4 depicts the multiple functional groups that provide multipleassay format detection of enzymatic activity. The terminal amino groupon B² is capable of forming covalent bonds with carboxylic acids onsolid supports. The presence of the quaternary ammonium on B³ provides apermanently charged moiety such that subsequent ionization is notrequired for analysis by MS/MS providing a robust and easily detectableand quantifiable detection of lysosomal enzyme activity.

FIG. 5 demonstrates a generic enzymatic reaction using an inventivesubstrate. Upon specific affinity binding and enzymatic reaction, thesubstrate is cleaved into two groups, a sugar moiety A and an aliphaticgroup B. The group B is optionally composed of a nitrophenyl, a lysine,a carnitinyl, and long-chain alkyl moieties. Both groups are optionallythen analyzed by MS/MS. An internal standard is also concurrentlysubject to the MS/MS analysis. The internal standard is an isotopicallylabeled analog of B with deuterium to replace hydrogen atom(s) on amethyl group. Alternatively, the deprotected terminal amino group on B²is reactive with a solid support such that the presence or absence ofproduct is optionally detected using immunoassay methods. Thedeprotected terminal amine can also be derivatized with a fluorescentreagent for HPLC, or other suitable detection method.

FIG. 6 illustrates exemplary inventive methods depicting detection ofenzymatic reactions using mass spectrometry that are advantageous incomparison to prior art methods. The inventive method eliminates manysteps inherent with the use of non-polar substrates and their relativeinternal standards. More specifically, the inventive method no longerneeds steps to clean up non-polar solvent and or detergents theinvolvement of which is detrimental to mass spectrometry machinery.These steps include: the extraction of enzymatic products intochloroform by a liquid-liquid extraction step, the removal of thedetergents by a silica-gel separation step, and the step of solid-phaseextraction (SPE) process. These extra steps of the prior art addstatistical error. Since the inventive substrates in accordance with thecurrent invention are more polar in contrast to the prior art, polarsolvents illustratively including pure methanol or pure ethanol areoperative herein.

All reagents including the substrates, enzymatic products, and internalstandards can be optionally purified by reverse-phase HPLC andcharacterized by ESI-MS, either in an online HPLC-MS assay or offline bycollection of the appropriate fractions.

EXAMPLES Example 1

For each sample, a disk of 3 mm diameter is punched from the areas ofdried blood on a filter paper into a well of a 96-well microtiter plate.The blood disk is then incubated directly with an assay solutioncontaining substrates directed to the Fabry disease at a finalconcentration of 5 mmol/L and internal standards at a finalconcentration of 0.1 mmol/L. To the assay solution, a finalconcentration of 0.5 mol/L sodium acetate buffer is also added. Theassay mixture containing the blood disk is incubated for 15 to 24 hoursat 37° Celsius with orbital shaking (150 rpm) in a thermostatic airshaker. After the incubation period, an aliquot of pure methanol isadded to each tube or well to terminate the enzymatic reaction. Beforegoing into the mass spectrometer, the incubated reaction mixture isdiluted with pure methanol. For the mass spectrometry analysis, theelectrospray source is operated in positive mode, and the ions aredetected in parent-ion scan mode. The amount of enzymatic product iscalculated from the ion abundance ratio of the product to the internalstandard minus that of a blank.

Example 2

In an alternative embodiment the product of the reaction with theinventive substrates is quantified by immunoassay. Blood spotted onfilter paper is reconstituted in buffer to liberate the activecomponents. One or an array of inventive substrates are added to thereaction chamber and the reaction allowed to proceed overnight (˜14hours). The reaction is quenched by the addition of 6× volumeglycine/NaOH pH 10.4. A sample of each reaction is added to the wells ofa high-binding irradiated microtiter plate and incubated overnight toallow sufficient binding of the reaction product to the wells of theplate. A standard curve of product in similar buffer/sample is alsoadded to the plate to serve as a basis for quantitation. After completebinding to the surface of the plate, the wells are washed twice withphosphate buffered saline (PBS) by the use of a squirt bottle, platewasher, or any other automated or non-automated plate washing system.Any additional sites for protein binding are subsequently blocked by theaddition of a blocking agent illustratively including 3% bovine serumalbumin in PBS or any other synthetic or natural blocking agent known inthe art. The blocking agent is incubated for two hours at roomtemperature. The wells are washed 3× with PBS. The primary antibody(s)is then added to the wells to recognize and bind the remainingsubstrate, or the product. The antibody(s) is incubated in the wells forat least 2 hours. The plate is washed four times to remove unboundantibody. If the primary antibody is labeled the plate is used fordetection. Optionally, a labeled secondary antibody is placed in theplate wells and allowed to incubate for an additional 2 hours followedby washing 4 times and detection by the appropriate method such as by afluorescent or optical plate reader.

Example 3

In an alternative embodiment the product of the reaction with theinventive substrates is quantified by immunoassay. Blood spotted onfilter paper is reconstituted in buffer to liberate the activecomponents. One or an array of inventive substrates immobilized toencoded particles are added to the reaction chamber, preferably amicroplate well, and the reaction allowed to proceed overnight (˜14hours). A standard curve of enzyme in similar buffer/sample is alsoadded to separate sets of encoded particles to serve as a basis forquantitation. The reaction is quenched by the addition of 6× volumeglycine/NaOH pH 10.4. The primary antibody(s) is then added to the wellsto recognize and bind the remaining substrate, or the product. Theantibody(s) is incubated for at least 30 minutes. If the primaryantibody is labeled the assay is ready for detection. Optionally, alabeled secondary antibody is placed in the plate wells and allowed toincubate for an additional 30 minutes. Detection is accomplished a flowcytometer.

Example 4

The active amino group on the B² of a preferred embodiment is amenableto numerous labeling procedures. Among these, fluorescent labelingoffers one of the most powerful methods of detection as it providesexcellent sensitivity, ability to quantitate relative to a standard, andcan be combined with other substrates with other fluorophores in a panelassay for multiple lysosomal enzymes. In a representative example, theB² side chain amine is specifically labeled with fluoroisothiocyanate(FITC). Derivitization of the inventive substrate of FIG. 1 is performedby addition of a FICT molecule to the terminal amine of the B² group.The purified/lyophilized substrate is resuspended in 0.1 M sodiumbicarbonate buffer, pH 9.0 at a concentration of 5 mg/ml. Immediatelyprior to reaction with substrate, dissolve 5 mg of FITC dye in 0.5 ml ofDMSO in the dark. With gentle vortexing, add 0.1 ml of dye solution ofthe substrate solution and incubate for 1 hour at room temperature inthe dark. The free unreacted dye is removed by gel filtration on a10×300 mm Sephadex G column pre-equilibrated in phosphate bufferedsaline. Concentration of the final product is determined by massspectrometry or other method known in the art. The labeled substrate isoptionally concentrated and aliquoted for storage at −20° C. untilfurther use.

The labeled substrate is used in a reaction for the detection ofglucocerebrosidase activity and the detection of Gaucher's disease. Foreach patient or control sample, a disk of 3 mm diameter is punched fromthe areas of dried blood on a filter paper into a micro-centrifuge tubeor a well of a 96-well microtiter plate. The blood disk is thenincubated directly with an assay solution containing labeled inventivesubstrate at a final concentration of 5 μmol/L and internal standards ata final concentration of 0.1 mmol/L. The assay mixture containing theblood disk is incubated for 15 to 24 hours at 37° C. with orbitalshaking (150 rpm) in a thermostatic air shaker. After the incubationperiod, an aliquot of pure methanol is added to each tube or well toterminate the enzymatic reaction. A sample of the reaction is added to asecond tube containing a HPLC mobile phase (methanol:water:acetic acid,82:18:0.1 vol/vol/vol). A 20-μl aliquot of the quenched reactionsolution is separated on a 4.6×250-mm Symmetry C₁₈ reverse-phase HPLCcolumn (Waters, Milford, Mass.) isocratically, at a rate of 1.3 ml/minusing methanol:water:acetic acid at 82:18:0.1 vol/vol/vol as a mobilephase. Fluorescence intensity is continuously monitored using afluorescence detector (model L-7480; Hitachi, Naperville, Ill.) at amedium gain sensitivity. The amount of labeled product in the sample isdetermined by comparing the area of the peak to that of an externalstandard comprised of labeled product at a known concentration. Theconcentration of product in the reaction is readily determined and theactivity of glucocerebrosidase determined by dividing the molesproduct/per unit of reaction time.

Example 5

In an alternative embodiment the product of the reaction with theinventive substrates is quantified by immunoassay wherein a specificantibody targeted to the cleaved inventive substrate is employed. Bloodspotted on filter paper is resolubilized in buffer to liberate theactive components. One or an array of inventive substrates are added tothe reaction chamber and the reaction allowed to proceed overnight (˜14hours). The reaction is quenched by the addition of 6× volumeglycine/NaOH pH 10.4. A microtiter plate is prepared in advance bycoating with an antibody generated in a mouse that is specific for thenitrophenyl-aminoacyl B¹-B² moiety. Any additional sites for proteinbinding are subsequently blocked by the addition of a blocking agentillustratively including 3% bovine serum albumin in PBS or any othersynthetic or natural blocking agent known in the art. A sample of eachreaction is added to the wells of a microtiter plate and incubated fortwo hours to overnight to allow sufficient binding of the reactionproduct to the wells of the plate. A standard curve of control productin similar buffer/sample is also added to the plate to serve as a basisfor quantitation. After complete binding to the surface of the plate,the wells are washed twice with phosphate buffered saline (PBS) by theuse of a squirt bottle, plate washer, or any other automated ornon-automated plate washing system. The primary antibody(s) is thenadded to the wells to recognize and bind the product at an alternativeepitope. The antibody(s) is incubated in the wells for at least 2 hours.The plate is washed four times to remove unbound antibody. If theprimary antibody is labeled the plate is used for detection. Optionally,a labeled secondary antibody is placed in the plate wells and allowed toincubate for an additional 2 hours followed by washing 4 times anddetection by the appropriate method such as by a fluorescent or opticalplate reader.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to be completelyincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well-adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It will be apparent thatother embodiments exist and are encompassed within the spirit of theinvention as defined by the scope of the claims.

The invention claimed is:
 1. A molecule comprising the formula:B¹-B²-B³-B⁴  (I) where B¹ is a methylene or 4-aminophenol; B² is adiaminohexanoyl or an amidylaminoacyl said B² covalently bound to B¹ byan amide bond and to B³ by an amide bond; B³ includes a quaternaryammonium and is a carnitinyl; and B⁴ is an alkyl chain with 10-20carbons linked to B³ via an ester bond.
 2. The molecule of claim 1wherein B¹ is a 4-aminophenol.
 3. The molecule of claim 1 where B² is alysine.
 4. The molecule of claim 1 wherein B² is derivatized by theaddition of a fluorophore.
 5. The molecule of claim 1 wherein(B¹-B²-B³-B⁴) includes a stable secondary prevalence isotope of anelement.
 6. The molecule of claim 5 wherein said stable secondaryprevalence isotope in each occurrence is selected from the groupconsisting of ²D, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, ³¹P and ³⁴S.
 7. A method fordetecting enzymatic activity, comprising: contacting a sample containinga target enzyme with a substrate having the formula:A-B¹-B²-B³-B⁴ where A is a monosaccharide or a disaccharide, said Acovalently bound to said B¹, and wherein said B¹-B²-B³-B⁴ is themolecule of claim 1, under conditions wherein the target enzyme iscapable of acting on the substrate to produce an enzymatic product; anddetecting the amount of the enzymatic product.
 8. The method of claim 7wherein the B² of said enzymatic product is derivatized by the additionof a fluorophore.
 9. The method of claim 7 wherein said substrate islinked to a solid support.
 10. The method of claim 7 wherein saiddetecting is by detecting the amount of substrate remaining on the solidsupport.
 11. The method of claim 7 wherein said B¹-B²-B³-B⁴ includes astable secondary prevalence isotope of an element.
 12. The method ofclaim 11 wherein said stable secondary prevalence isotope in eachoccurrence is selected from the group consisting of ²D, ¹³C, ¹⁵N, ¹⁷O,¹⁸O, ³¹P and ³⁴S.